Heparin derivatives and methods for preparing same



United States Patent Oiiice 3,232,838 Patented Feb. 1, 1966 19 Claims. for. 167-74) The invention relates to the novel compounds, N-(mono or di-sulfobenzoyl)-N-desulfoheparins and their alkali metal salts, and to a novel process for preparing the novel compounds. The invention also relates to novel anticoagulant compositions and to a method of preventing the coagulation of blood.

Heparin is a known compound which possesses antilipemic and anti-coagulant activity. Various derivatives of heparin are also known which possess a strong antilipemic activity with a minimum of anti-coagulant activity. US. Patent No. 3,065,140 describes N-acylated derivatives of N-desulfoheparin, such as N-benzoyl-N-desulfoheparin, N-p-nitro-benzoyl-N-desulfoheparin and N-3,5- dimethylbenzoyl-N-desulfoheparin which have a lower anti-coagulant activity than heparin. US. Patent No. 3,033,751. describes substituted heparylurea derivatives prepared from N-desulfoheparin, such as n-butyl heparylurea, phenyl heparylurea and ot-naphthyl heparlurea which possess a greater ratio of antilipemic activity to anti-coagulant activity than heparin. The compounds of the invention possess a clear, more prolonged anti-coagulant activity than heparin.

It is an object of the invention to provide novel N- (sulfobenzoyl)-N-desulfoheparins and their alkali metal salts.

It is another object of the invention to provide a novel process for the preparation of N-(sulfobenzoyl)-N-desulfoheparins and their alkali metal salts.

It is another object of the invention to provide novel anti-coagulant compositions having a prolonged activity.

It is a further object of the invention to provide a novel method of preventing the coagulation of blood.

These and other objects and advantages of the invention will become obvious from the following detailed description.

The novel compounds of the invention are N-(monoanddi-sulfobenzoyl)-N-desulfoheparins and their alkali metal salts which have the probable structural formula wherein R is selected from the group consisting of hydrogen and an alkali metal and 1 to 2 of R R R and R are sulfonic acid groups in the free or alkali metal salt form and the rest of R R R and R being hydrogen and rt is about 10.

The novel process of the invention for the preparation of N-(monoand di-sulfobenzoyl)-N-desulfoheparin cornprises reacting N-desulfoheparin with a high moiecular weight quaternary ammonium salt to form the corresponding quaternary ammonium salt of N-desulfohepar-in, reacting the latter with an acid anhydride selected from the group consisting of an anhydride of a mono-suitobenzoic acid, an anhydride of a di-sulfobenzoic acid, a mixed anhydride of a mono-sulfobenzoic acid and a lower I R2 raglan alkanoic acid and a mixed anhydride of a di-sulfobenzoic acid and a lower alkanoic acid to form the: corresponding N-(sulfobenzoyl)-N-desultoheparin which may be recovered or reacted with an alkali metal salt of a lower alkanoic acid to form the corresponding alkali metal salt of the N-(sulfobenzoyl)-N-desulfoheparin.

The high molecular weight quaternary ammonium salts which are reacted with the N-desulfoheparin are known. Examples of suitable salts are Hyamine 2389 which is a trirnethyl (methyl dodecyl benzyl) ammonium chloride, Arquad 2C which is dilauryl dimethyl ammonium chloride, and preferably Hyamine 1622 which is benzyldimethyl 2 [Z-(p-1,1,3,3-tetramethyl butylphenoxy)-ethoxy] ethyl ammonium chloride.

Examples of suitable anhydrides and mixed anhydrides of the sulfobenzoic acids useful in the process are o-sulfobenzoic acid anhydride, 2,4-d-isulfobenzoic acid anhydride, the mixed anhydride of 3,5-disulfobenzoic acid and ethoxy carbonic acid and the mixed anhydride of rn-sulfobenzoic acid and ethoxyearbonic acid.

The novel anti-coagulant compositions of the invention are comprised of a compound selected from the group consisting of N-(monoand di-sulfobenzoyD-N- desulfoheparins and their alkali metal salts and a major amount of a pharmacological carrier, preferably a liquid injectable carrier. The compositions possess a clear, prolonged anti-coagulant activity which lasts for a period of 6 to 10 hours from the time of administration.

The N-(monoand di-sulfobenzoyl)-N-desulfoheparins of the invention have a more prolonged period of activity than heparin and avoids the repeated intravenous injections or continuous venous perfusions required for very high doses of heparin. They also act with more rapidity for a well determined period of time than anti-coagulant H derivatives of. dicoumarine which inhibit the synthesis of thrombin in the liver (antagonists of vitamin K) and, therefore, have a retarding activity but whose duration cannot be absolutely determined beforehand. Moreover, the activity of the compounds of the invention occurs in all stages of coagulation and, therefore, their anti-coagulant activity is more certain and less dangerous for the organism.

The novel method of preventing the coagulation of blood comprises intravenously administering an effective amount of a compound selected from the group consisting of N-(monoand di-sulfobenzoyl)-N-desulfoheparin. The usual useful dosage is 400 mg. to 2 gm. per day depending on the method of administration.

COOR cHao osR in the following examples there are described several preferred embodiments to illustrate the invention. However, it is to be understood that the invention is not intended to be limited to the specific embodiments.

EXAMPLE I Preparation of the sodium salt of N-(2-sztlfobenz0yl)- N-desulfoheparin 9.07 g. of N-desulfoheparin were introduced into 50 cc. of water and then 250 cc. of an aqueous solution containing 10% of Hyamine 1622 were added. The precipitate formed was homogenized and after the reaction mixture was allowed to remain at rest for a period of about one hour, it was vacuum filtered and the precipitate washed with water. 28.5 g. of the Hyamine salt of N- desulfoheparin were obtained which was used as such for the following step of the synthesis.

3 g. of the said Hyamine salt were introduced under agitation into 60 cc. of tetrahydrofuran and agitated until solution occurred. Then, successively and under agitation, 3 cc. of triethylamine and 1.5 g. of o-sulfobenzoic acid anhydride were added. The agitation was continued for a period of hours at room temperature and the solution was then evaporated to dryness under vacuum. The residue was taken up with cc. of aqueous n-buta- 1101 and the n-butanol solution was extracted several times with a 20% solution of sodium acetate. The combined extracts were filtered and after the filtrate was poured into methanol, the solution was allowed to stand at rest for a period of one hour. The precipitate formed was vacu um filtered, washed with methanol and dried to obtain 1.047 g. of the sodium salt of N-(2-sulfobenzoyl)-N-de- 'sulfoheparin.

U.V. spectra.(N/l0 hydrochloric acid) absorption at 266 and 272 m corresponding to 1.3*1.4 10- mols of o-sulfobenzoic acid per gram. This product was soluble in water and insoluble in alcohol, ether, acetone, benzene and chloroform. Dilute aqueous acids hydrolyzed it and dilute aqueous alkalis degraded it.

AIZalySl S.-(c7 HqqOq7N S 1Na15) (2,976.12) culated: S, 11.85%. Found: 11.4.

Anticoagulant activity.3 :1.5 ATU (anti-thrombic units) mg.

This compound is not described in the literature.

EXAMPLE 11 Preparation of the sodium salt of N-(2,4-disul/obenzoyl)- N-dcsulfoheparin STEP A.PREPARATION OF THE HYAMINE 1622 SALT OF N-DESULFOI-IEPARIN 17.88 g. of the potassium salt of N-desulfoheparin were dissolved in 100 cc. of water and then 500 cc. of an aqueous solution of 10% Hyamine 1622 was added thereto. The reaction mixture was homogenized and then allowed to stand at rest for a period of about 1 to 2 hours. The precipitate formed was vacuum filtered, washed in water and dried under vacuum to obtain 56 g. of the Hyamine 1622 salt of N-desulfoheparin (compound A).

STEP B.PREPARATION OF HYAMINE 1622 SALT OF THE ANHYDRIDE OF 2,4-DISULFOBENZOIC ACID g. of the sodium salt of 2,4-disulfobenzoic acid were dissolved in 700 cc. of water and 70 cc. of formic acid and then 1,550 cc. of an aqueous solution of 10% Hyamine 1622 were added. The resulting solution was extracted several times with methylene chloride. The extracts were combined and evaporated, water was removed by entrainment with benzene. The dry residue was taken up with 700 cc. of trichloroethylene and the mixture was heated to reflux until solution occurred. Then the solution was allowed to cool to 50 to 60 C. and 105 cc. of thionyl chloride was introduced under agitation. The agitation was continued for about 2 hours at a temperature situated between and C. Then distillation of the solution was started under normal pressure, and continued under vacuum until there remained only traces of thionyl chloride in the mixture. The residue was taken up with 700 cc. of tetrahydroturan and the resulting solution was treated with animal carbon black, filtered and a tetrahydrofuran solution of the Hyamine 1622 salt of the anhydride of 2,4-disulfobenzoic acid (compound B) was obtained which was used as such for the following step of the synthesis.

,STEP C.PREPARATION OF THE SODIUM SALT OF N- (2,4-DISULFOBENZOYL) -N-DESULFOHEPARIN 49 g. of the Hyamine 1622 salt of N-desulfoheparin (A) were introduced under agitation in 1000 cc. of tetrahydrofuran and after solution occurred at room temper.-

ture, solution B prepared in Step B was added, and the reaction mixture was subjected to agitation for a period of about 17 hours at room temperature. Then the reaction mixture was evaporated to dryness under vacuum at temperatures below 50 C. The residue was taken up with 500 cc. of aqueous n-butanol and extracted several times with a 20% aqueous solution of sodium acetate. The extracts were combined, filtered, and 750 cc. of methanol were added thereto. The mixture was allowed to stand for about 1 to 2 hours and the precipitate formed was filtered. The precipitate was washed successively with methanol and ether and dried to obtain the raw sodium salt of N-(2,4-disulfobenzoyl)-N-desulfoheparin.

This product was purified by passing an aqueous solution of the said salt through .a column of a strongly basic anion exchange resin, such as that possessing the functional structure of a quaternary ammonium group and recovering the traction giving a positive test of precipitation of Hyamine 1622. The column was rinsed with distilled water and the eluates were combined, treated with animal carbon black and filtered. The filtrate was neutralized by the addition of acetic acid and the pH was then adjusted to 7.8 with a sodium hydroxide solution. The said salt was precipitated by the addition of methanol and the precipitate was separated by vacuum filtration, was washed successively with methanol and ether, and dried to obtain 16.7 g. of the sodium salt of N-(2,4-d.isulfobenzoyl -N-desulfoheparin.

The product was soluble in water and insoluble in alcohol, ether, acetone, benzene and chloroform.

Sulphur content: 12.65% (theory: 14.18%).

U.V. spectra.(ln N/ 10 hydrochloride acid solution).

Anticoagulant activity.-66.2 ATU/mg. This compound is not described in the literature.

EXAMPLE III Preparation 0 the sodium salt of N-(3,5-disulfobenz0yl)- N -d esul yoh eparl'n STEP A.--PREPARATION OF THE MIXED ANHYDRIDE OF 3,5-DISULFOBENZOIC ACID AND ETHOXYCAR- BONIC ACID In a 500 cc. conical flask, 3.75 g. of the potassium salt of 3,5-disulfobenzoic acid were dissolved in 50 cc. of distilled water and the pH of the solution was adjusted to 10 by addition of potassium hydroxide. 160 cc. of a 10% solution of Hyamine 1622 were then added and the Hyamine 1622 salt of 3,5-disulfobenzoic acid which precipitated was recovered by filtration. The filter cake was then extracted twice with cc. aliquots of methylene chloride and the extracts were washed with water, dried over sodium sulfate and evaporated to dryness. The residue was redissolved in 30 cc. of tetrahydrofuran and the solution was transferred into a three-necked balloon flask and cooled to between 0 and 5 C. under agitation and an atmosphere of nitrogen. The pH was adjusted to 10 by the addition of 0.1 cc. of triethylamine and then 0.75 cc. of ethylchloroformate were added while agitation was maintained under an atmosphere of nitrogen. At the end of 15 minutes, 1.5 cc. of a 10% solution of triethylamine formate in tetrahydrofuran were added to decompose the excess of ethylchloroformate. There was thus obtained a solution of the mixed anhydride of 3,5 -disulfobenzoic acid and formic acid.

STEP B.IREPARATION OF N-(3,5-DISULFOBENZOYL)- N-DESULFOHEPARIN To the mixed anhydride solution prepared in Step A there was added a solution of 2 gm. of the Hyarnine 1622 salt of N-desulfoheparin, 30 cc. of tetrahydrofuran and 0.5 cc. of triethylamine. The resulting solution was stirred while bubbling in a current of nitrogen and was then evaporated to dryness under vacuum to form an amorphous residue of N-(3,5-disulfobenzoyl) -N-desulfoheparin.

6 distilled ethylchloroformate was introduced and the temperature of the reaction media mounted slowly and then returned to 5 C. The mixture was maintained for 30 minutes at this temperature and then 3 cc. otf a 10% solu- STEP C 'QREPA,RATION 0F TIIE SODIUBf SALT 0F 5 tion of triethylamineformate in tetrahydrofuran Were N-(3,5-DISULFO-BENZOYL')-N-DESULFOHEPARIN added to obtain a solution of the mixed anhydride of m- The residue obtained in Step B Was dissolved in 30 sulfcbenzoic acid and formic acidcc. of butanol saturated with water and this solution was STEP C' PREPARATION OF THE SODIUM SALT OF extracted three times with 5 cc. aliquots of a aqueous N-(B-SULFOBENZOY-L nmnsum onnrnum solution of Sodium 'f The q u solution W33 10 cc. of a tetrahydrofuran solution containing 10% of filtered and P0111'ed 1nt0 100 of methanolh the Hyamine 1622 salt of N-desulfo-heparin were added dh Salt of N(3s'dlsulfobenzoyl)'N'desulf9hepann to the solution of the mixed anhydride of m-sulfobenzoic Preclpltatei T1115 Salt Was recovered y filtratlon, Was acid and formic acid. The container which contained Vacuum filtefedi Washefi with methanol containing 2 the Hyamine 1622 salt of N-desulfoheparin was rinsed Water and qlned to Obtam O5 to Y 15 with 10 cc. of tetrahydrofuran and the rinse liquids were of the sodium salt of N-(3,5-disul fobenzoyl)-Ndcsulf poured into the reaction mixture. The mixture was heparin. The product had an amidification value varying agitated f a period of one hour at 0 C. under an betwee 82 o 95 mosphere of nitrogen and the reaction mixture was then U.V. speclIa.--(N/ 1O hydrochloric acid) inflect at evaporated to dryness under vacuum. The amorphous about 270 and 230 M indicating the lfm of to 20 residue of N-(m-sulfobenzoyl)-N-desulfohep-arin was re- 1-13'X10 3 mols of 3i5'dlsuifobenzolc 301d P gram dissolved in 50 cc. of butanol saturated with water. The equwzElgnt of pmduct Ktheoreficah 118x 10%/g); solution of N-(rn-sulfobenzoyl) N desulfoheparin was AltlWagmam ATU/mg- Thls then extracted with 10 cc., again 10 cc. and finally 5 cc. un 15 not descrlbed 111 the htemmreof a 20% aqueous solution of sodium acetate. The aque- XA L 1 0 ous pllzses weirfe COIl11'1bini3d,TdCaIlt6d, filtered and poured into cc. 0 met ano. e reci itate was set arated Prepmmlo" of the Sodium salt N'w'sulfobenzoyn' by filtration, vacuum filtered, wa shed and dried to obtain Ndmlfohelmm 1.05 g. yield) of the sodium salt of N-(3-sulfo- STEP A.-PREP1XRATION 013 This HYAMINE 162a SALT 30 rb oyl)-N-desu1foheparin.

0E M'SULFOBENAOJIC ACID Analysis.-Percent NH 4% (expressed in N-desul- Into a 300 cc. Erlenmeyer flask there were introduced foheparin). Percent S, 11.5% (theoretical: 11.88%). successively 5.2 g. of sodium m-sulfobenzoate, 125 cc. of UV, peclra,-(N/ 1O hydrochloric acid): distilled water and 2.5 cc. of sodium hydroxide solution. l fi fi The sodium m-sul fobenzoate dissolved rapidly and an alkaline solution having a pH in the neighborhood of 10 00 230 E} was thus obtained. 220 cc. of a 10% aqueous solution of 270 mu i Zn.= hyamine 1622 were then added and the m-sulfobenzoate of Hyamine 1622 precipitated as an amorphous precipitate. Thls absiorptlon corresponds "E 1'O6X1073 Incl of After the precipitate was separated by filtration it was 40 iobenzolc acld E gramieqmYalent 9 producbuheoret extracted twice with 100 cc. aliquots of methylene chloride malt 135x10 and the amldlficatlon value 18 thus of and the organic extracts were combined, washed with the Order of water, dried over sodium sulfate and evaporated to dry- Anjfwoagula' U mess at atmospheric pmssura The dry residue of the This compound is not .descnbed 1n the literature. H amine 1622 salt of m-sul-fobenzoic acid was redissolved in about 50 cc. of tetrahydrofuran. A fter complete solu- PHARMACOLOGICAL DATA tion the solution was brought to an exact volume of cc. Coagulant retardation activity by the addition of more tetrahydrofuran. I STEP B.PREPARATION OF THE MIXED ANHYDRIDE The antlcoagulant acimty of 'i and dlsulfo" on M-SULFOBENZOIC ACID AND 'ETHOXY oamsomc benzoyl)-N-desulfoheparms of the invention was com- ACID pared with heparin on rabbits. The compounds tested Into a balloon flask having three necks, 50 cc. of the were administered intravenously by injection to the rabbits tetrahydrofuran solution of the Hyamine 1622 salt of at doses of 10 mg./kg. and 20 mg./kg. Blood samples m-sulfobenzoic acid prepared in Step A were introduced. were taken at regular intervals 2, 4, 6, 8 and 10 hours The mixture was cooled to 5 Oil under agitation and after the administration of the compounds and the time of under atmosphere of nitrogen and the pH of the solution coagulation of the blood determined. The results are was adjusted to 10 with triethylamine. Then 1 cc. of resummarized in Table I.

TABLE I Anticoagulant Activity 10 mgJkg. 20 mgjkg. Compound Administered Number of Duration Number of Duration IU/kg. of action IU/kg. of action in hours in hours Heparin, sodium salt 1, 350 4 2, 700 6-7 N-(Q-sulfobenzoyl)-N-desulioheparin,

sodium salt 340 6 680 9 N-(3-sulfobenz0 N-desulfoheparin,

sodium salt -250 3 N-(3,5-disulfobenzoyl)-N-desulfoheparin, sodium salt -300 2 -600 3 N-(2,4-di$u1fohenz0yl)-N-desulfoheparin, sodium salt 220 6-7 440 9-10 As can be seen from Table I, the N-(sulfobenzoyl)-N- desulfoheparins of the invention possess a more pro longed anti-coagulant activity at anti-coagulant dosages (expressed in International Units) infinitely less than those of heparin necessary to obtain an effect of the same order.

Various modifications of the process and compositions of the invention may be made without departing from the spirit or scope thereof, and it should be understood that the invention is intended to be limited only as defined in the appended claims.

We claim:

1. A compound selected from the group consisting of N-(mono-sulfobenzoyl)-N-desulfoheparin and N-(di-sulfiobenzoyl)-N-desul-foheparin and their alkali metal salts.

2. N-(Z-sulfobenzoyl)-N-desulfoheparin.

3. The sodium salt of N-(Z-sulfobenzoyl)-N-desulfoheparin.

4. N(2,4-disulfobenzoyl)-N-desulfoheparin.

5. The sodium salt of N-(2,4-disulfobenzoyl)-N-desul foheparin.

6. N- 3,5 -disulfobenzoy1 -N-desulfoheparin.

7. The sodium salt of N-(3,5-disulfobenzoyl)-N-de- 8. N-(3-sul-fobenzoyl)-N-desulfoheparin. sulfoheparin.

9. The sodium salt of N-(3-sulfobenz0yl)-N-desulfoheparin.

10. A process for the preparation of a compound selected from the group consisting of N- (mono-sulfo- :benzoyl)-N-desulfoheparin and N-(di sulfobenzoyl) N- desulfoheparin which comprises reacting N-desulfoheparin with a high molecular Weight quaternary ammonium salt to form the corresponding quaternary ammonium salt of N-desulfoheparin, reacting the latter with an acid anhydride selected from the group consisting of anhydrides of mono-sulfobenzoic acid and disulfobenzoic acid and mixed anhydrides of mono-sulfobenzoic acid and di-sulfobenzoic acid with a lower alkanoic acid to form the desired N- (sulfobenzoyl)-N-desulf0heparin.

11. The process of claim 10 wherein the N-(sulfobenzoyl)-N-desulfoheparin is reacted with an alkali metal salt of a lower alkanoic acid to form the corresponding alkali metal salt of the N-(sulfobenzoyl)-N-desulfoheparm.

12. The process of claim 10 wherein the acid anhydride is the mixed anhydride of m-sulfobenzoic acid and ethoxycarbonic acid.

13. The process of claim 10 wherein the high molecular weight quaternary ammonium salt is benzyl dimethyl-Z- [Z-(p-l,1,3,3,-tctramethyl-butyl-phenoxy) ethoxy] ethyl ammonium chloride.

14. The process of claim 11 wherein the alkali metal salt of a lower alkanoic acid is sodium acetate.

15. Anti-coagulant compositions comprising a compound selected from the group consisting of N-(rnonosulfobenzoyl) -N-desulfohep arin and N- (d-i-sulf-obenzoyl N-desulfoheparin and their alkali metal salts and a major amount of a pharmaceutical carrier.

16. Anti-coagulant compositions comprising a compound selected from the group consisting of N-(monosulfobenzoyl)-N-desulfoheparin and N-(di-sulfobenzoyD- N-desulfoheparin and their alkali metals salts and a major amount of a liquid, injecta ble carrier.

1'7. The process of claim 10 wherein the acid anhydride is o-sulfobenzoic acid anhydride.

18. The process of claim 10 wherein the acid anhydride is 2,4-disul'f0benzoic acid anhydride.

19. The process of claim 10 wherein the acid anhydride is the mixed anhydride of 3,5-disulfobenzoic acid and ethoxycarbonic acid.

References Cited by the Examiner UNITED STATES PATENTS 3,118,817 1/1964 Nomine 167-74 JULIAN S. LEVITT, Primary Examiner.

FRANK CACCIAPAGLIA, 111., Examiner. 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF N-(MONO-SULFOBENZOYL)-N-DESULFOHEPARIN AND N-(DI-SULFOBENZOYL)-N-DESULFOHEPARIN AND THEIR ALKALI METAL SALTS. 